DE10014546A1 - Nucleotide sequences encoding the dapC gene and methods of producing L-lysine - Google Patents

Abstract

The invention relates to an isolated polynucleotide from coryneform bacteria containing at least one polynucleotide sequence selected from the group DOLLAR A a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide having the amino acid sequence according to SEQ ID NO. Contains 2, DOLLAR A b) polynucleotide encoding a polypeptide containing an amino acid sequence which are at least 70% identical to the amino acid sequence of SEQ ID NO. 2, DOLLAR A c) polynucleotide complementary to the polynucleotides of a) or b), or DOLLAR A d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c), DOLLAR A and a method for the fermentative production of L-amino acids, in particular L-lysine which is characterized in that in the coryneform microorganisms which in particular already produce L-amino acid, DOLLAR A a) at least amplifies, in particular overexpresses, the nucleotide sequence coding for the dapC gene, DOLLAR A b) enriches the desired L-amino acid in the medium or in the cells of the bacteria and DOLLAR A c) isolated the desired L-amino acid.

Description

The invention relates to coding for the dapC gene Nucleotide sequences and methods for fermentative Production of L-lysine using coryneforms Bacteria in which the dapC gene (N-succinyl- Aminoketopimelate transaminase gene), in particular overexpressed.

State of the art

Amino acids, especially L-lysine, find in the Human medicine and in the pharmaceutical industry, but especially in animal nutrition, application.

It is known that amino acids by fermentation of Strains coryneformer bacteria, in particular Corynebacterium glutamicum. Because of the Great importance is constantly attached to the improvement of Manufacturing process worked. Process improvements can fermentation measures such. B. stirring and Supply of oxygen, or the composition of the Nutrient media such. B. the sugar concentration during the Fermentation, or the work-up to product form by z. As ion exchange chromatography or the intrinsic Performance characteristics of the microorganism itself affect.

To improve the performance characteristics of this Microorganisms become methods of mutagenesis, selection and mutant selection applied. In this way you get Strains that are resistant to antimetabolites such. B. the Lysine analog S- (2-aminoethyl) -cysteine or auxotrophic for are regulatory metabolites and L-amino acids such as B. produce L-lysine.

For some years now also methods of Recombinant DNA technology for strain improvement amino acid producing strains of Corynebacterium used by  one amplifies individual amino acid biosynthesis genes and investigated the effect on amino acid production. Reviews for this can be found among others Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6: 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).  

Object of the invention

The inventors have set themselves the task of new Measures for the improved fermentative production of Lysine, provide.

Description of the invention

L-lysine is found in human medicine, in the pharmaceutical Industry and especially in animal nutrition application. There is therefore a general interest in new ones improved process for the production of L-lysine, provide.

If in the following L-lysine or lysine are mentioned, are so that not only the base, but also the salts such. B. Lysine monohydrochloride or lysine sulfate.

The invention relates to an isolated polynucleotide from coryneform bacteria containing at least one polynucleotide sequence selected from the group

• a) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, the the amino acid sequence of SEQ ID no. 2 contains
• b) polynucleotide encoding a polypeptide having a Contains at least 70% of the amino acid sequence is identical to the amino acid sequence of SEQ ID NO. 2,
• c) polynucleotide that is complementary to Polynucleotides of a) or b), or
• d) polynucleotide containing at least 15 successive nucleotides of Polynucleotide sequences of a), b) or c).

The invention likewise provides the polynucleotide according to claim 1, which is preferably a replicable DNA comprising:

• a) the nucleotide sequence shown in SEQ ID no. 1, or
• b) at least one sequence corresponding to the sequence (i) within the range of degeneration of the corresponds to genetic codes, or
• c) at least one sequence identical to that for the sequence (i) or (ii) complementary sequence hybridized, and optionally
• d) functionally neutral sense mutations in (i).

Other items are
a polynucleotide according to claim 4, containing the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide encoding a polypeptide having the amino acid sequence as set forth in SEQ. ID. 2, contains,
a vector containing the polynucleotide according to claim 1, in particular a shuttle vector or the plasmid vector pXT-dapCexp, which is shown in FIG. 2 and deposited under DSM 13254 in DSM 5715
and coryneform bacteria containing the vector as the host cell.

The invention also relates to polynucleotides which are in consist essentially of a polynucleotide sequence, the are available by screening by hybridization a corresponding gene bank containing the complete gene the polynucleotide sequence according to SEQ ID no. 1 contain, with a probe, the sequence of said Polynucleotide according to SEQ ID no. 1 or a fragment thereof contains and isolation of said DNA sequence.

Polynucleotide sequences according to the invention are as Hybridization probes suitable for RNA, cDNA and DNA to to isolate full length cDNA, the N-succinyl  Aminoketopimelat transaminase encode and such cDNA or Isolate genes that have a high similarity of sequence with the N-succinyl-aminoketopimelate transaminase gene respectively.

Polynucleotide sequences according to the invention are further suitable as a primer for the production of DNA from genes that encode N-succinyl-aminoketopimelate transaminase, by the polymerase chain reaction (PCR).

Such as probes or primers serving oligonucleotides contain at least 30, preferably at least 20, completely more preferably at least 15 consecutive Nucleotides. Also suitable are oligonucleotides with a length of at least 40 or 50 nucleotides.

"Isolated" means from its natural environment separated out.

"Polynucleotide" generally refers to Polyribonucleotides and polydeoxyribonucleotides, wherein it unmodified RNA or DNA or modified RNA or DNA can act.

By "polypeptides" is meant peptides or proteins, the two or more linked via peptide bonds Contain amino acids.

The polypeptides of the invention include a polypeptide according to SEQ ID no. 2, in particular those with the biological activity of the N-succinyl-aminoketopimelate Transaminase and also those which account for at least 70% are identical to the polypeptide according to SEQ ID no. 2, preferably at least 80% and especially at least 90% to 95% identity with the polypeptide according to SEQ ID no. 2 and have the said activity.

The invention further relates to a method for fermentative production of amino acids, in particular L- Lysine, using coryneform bacteria, which in particular already produce an amino acid, and in  those coding for the dapC gene nucleotide sequences amplified, in particular overexpressed.

The term "reinforcement" describes in this context the increase in intracellular activity of one or of several enzymes in a microorganism that are caused by the corresponding DNA are encoded by, for example the copy number of the gene (s) increases, a strong Promoter used or used for a gene corresponding enzyme with a high activity and coded if necessary, combine these measures.

The microorganisms that are the subject of the present Invention, L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, Molasses, starch, cellulose or glycerol and ethanol produce. It may be representative coryneformer Bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum to call in the professional world known for their ability to use L-amino acids to produce.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are the wild-type strains known, for example
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium molassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-lysine producing mutants or strains produced therefrom, such as
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464
Corynebacterium glutamicum DSM5715
Corynebacterium glutamicum DSM12866 and
Corynebacterium glutamicum DM58-1.

The inventors were able to use the new, for the enzyme N- Succinyl aminoketopimelate transaminase gene (EC 2.6.1.17) to isolate the coding dapC gene of C. glutamicum.

For isolation of the dapC gene or other genes of C. glutamicum will initially be a gene bank of this Microorganism created in E. coli. The creation of Genebanks is in well-known textbooks and Written down manuals. As an example, be that Textbook by Winnacker: Genes and Clones, An Introduction to genetic engineering (Verlag Chemie, Weinheim, Germany, 1990) or the handbook of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is the E. coli K-12 strain W3110, described by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of C. glutamicum ATCC13032, the with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) again describe a gene bank of C. glutamicum ATCC13032 using the cosmid pHC79 (scab and Collins, Gene 11, 291-298 (1980)). For the production of a Genbank of C. glutamicum in E. coli can also use plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) become. As hosts, such E. coli are particularly suitable.  Strains which are restriction and recombination defective. An example of this is the strain DH5αmcr, by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The help of cosmids cloned long DNA fragments can then again in common, for sequencing subcloned suitable vectors and then sequenced be, as it is z. In Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977).

In this way, the new coding for the gene dapC DNA sequence of C. glutamicum obtained as SEQ ID NO Part of the present invention is. Was continued from the present DNA sequence with those described above Methods the amino acid sequence of the corresponding protein derived. In SEQ ID NO 2 is the resulting Amino acid sequence of the dapC gene product shown.

Coding DNA sequences consisting of SEQ ID NO 1 by the degeneracy of the genetic code are also part of the invention. In the same way DNA sequences encoding SEQ ID NO 1 or parts of SEQ ID NO 1 hybridize, part of the invention. In the Experts are still conservative amino acid exchanges such as B. Exchange of glycine for alanine or of Aspartic acid against glutamic acid in proteins as "Sense mutations" known to none fundamental change in the activity of the protein lead, d. H. are functionally neutral. It is also known that changes at the N- and / or C-terminus of a protein its function does not significantly affect or even can stabilize. Information on this is the expert among others Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), O'Regan et al. (Gene 77: 237-251 (1989)); Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)); Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in known textbooks of Genetics and molecular biology. Amino acid sequences that  correspondingly from SEQ ID NO 2, are also part of the invention.

In the same way, DNA sequences that are identical to SEQ ID NO. 1 or parts of SEQ ID NO 1 hybridize part of the Invention. Finally, DNA sequences are part of Invention produced by the polymerase chain reaction (PCR) be made using primers that are from SEQ ID NO 1 revealed. Have such oligonucleotides typically at least 15 nucleotides in length.

Instructions for the identification of DNA sequences by means of Hybridization is found among others in the art Manual "The DIG System Users Guide for Filters Hybridization "of the company Roche Diagnostics GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). Instructions for the amplification of DNA sequences using the polymerase chain reaction (PCR) will be apparent to those skilled in the art among other things in the handbook of Gait: Oligonukleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Academic Spectrum Verlag, Heidelberg, Germany, 1994).

The inventors found that coryneform bacteria after Overexpression of the dapC gene in an improved manner L-lysine, to produce.

To achieve overexpression, the copy number of the corresponding genes are increased, or it may be the Promoter and regulatory region or the Ribosome binding site located upstream of the Structural gene is mutated. In the same way act expression cassettes upstream of the Structural gene to be installed. By inducible Promoters it is additionally possible to express in the Progress of fermentative L-lysine production increase. By measures to extend the lifetime of the m-RNA the expression is also improved. Continue by preventing degradation of the enzyme protein as well  enhances the enzyme activity. The genes or gene constructs can either be in different plasmids Copy number present or integrated in the chromosome and be amplified. Alternatively, a further Overexpression of the relevant genes by altering the Media composition and culture leadership can be achieved.

Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), to Eikmanns et al. (Gene 102, 93-98 (1991)), in the European Patent Specification EPS 0 472 869, in US Pat. No. 4,601,893 Schwarzer and Puhler (Bio / Technology 9, 84-87 (1991)) Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246 to Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese Patent Laid-Open Publication JP-A-10-229891 Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), by Makrides (Microbiological Reviews 60: 512-538 (1996)) and in known textbooks of genetics and molecular biology.

By way of example, the dapC gene according to the invention was obtained with the aid of of plasmids overexpressed.

Suitable plasmids are those which are in coryneform Bacteria are replicated. Many well-known Plasmid vectors such. PZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other Plasmid vectors such. For example, those based on pCG4 (US-A 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (US Pat. No. 5,158,891) can be used in the same way become.  

An example of a plasmid by means of which the dapC gene can be overexpressed is the E.coli-C.glutamicum shuttle vector pXT-dapCexp. It contains the replication region rep of the plasmid pGA1 including the replication effector per (US Pat. No. 5,175,108, Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetracycline-resistance-mediating tetA (Z) gene of the plasmid pAG1 (FIG. Genbank entry to the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) with the accession number AF121000), as well as the origin of replication, the trc promoter, the termination regions T1 and T2, and the lacI ^q - Gene (repressor of the lac operon of E. coli) of the plasmid pTRC99A (Amann et al., (1988), Gene 69: 301-315).

The pendulum vector pXT-dapCexp is shown in FIG .

Furthermore, such plasmid vectors are suitable with the aid of one of the procedure of gene amplification by Integration into the chromosome can apply, just like it for example, Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) Duplication or amplification of the hom-thrB operon has been described. This method becomes the complete one Gene is cloned into a plasmid vector which is in a host (typically E. coli), but not in C. glutamicum can replicate. Suitable vectors are, for example, pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994) Journal of Biological Chemistry 269: 32678-84; US Pat. No. 5,487,993), pCR® Blunt (Invitrogen, Groningen, the Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (shrink et al. 1991, Journal of Bacteriology 173: 4510-4516) Question. The plasmid vector containing the gene to be amplified is subsequently by conjugation or Transformation into the desired strain of C. glutamicum transferred. The method of conjugation is for example  in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for Transformation are, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross over" event contains the resulting strain at least two copies of the relevant Gene.

Additionally, it may be responsible for the production of L-lysine be advantageous, in addition to the dapC gene one or more Enzymes of the respective biosynthetic pathway, glycolysis, the To amplify anaplerotic or amino acid export or to overexpress.

Thus, for example, in addition to the dapC gene for the production of L-lysine, one or more of the genes selected from the group may be simultaneously

• - that for a feed back resistant aspartate kinase coding lysC gene (Kalinowski et al., (1990), Molecular and General Genetics 224: 317-324),
• - that for the aspartate-semialdehyde dehydrogenase coding asd gene (EP-A 0 219 027, Kalinowski et al. (1991), Molecular Microbiology 5: 1197-1204, and Kalinowski et al. (1991), Molecular and General Genetics 224: 317-324),
• The dapA- coding for the dihydrodipicolinate synthase Gen (EP-B 0 197 335),
• The dapE- coding for the dihydrodipicolinate reductase Gen (Genbank entry accession number X67737; Pisabarro et al. (1993), Journal of Bacteriology, 175 (9): 2743-2749),
• - The coding for tetrahydrodipicolinate succinylase dapD gene (Genbank entry accession number AJ004934;  Wehrmann et al. (1998), Journal of Bacteriology 180: 3159-3163),
• - that for N-succinyl-diaminopimelate-desuccinylase coding dapE gene (Genbank entry accession number X81379; Wehrmann et al. (1994), Microbiology 140: 3349- 3356),
• The dapE gene encoding diaminopimelate epimerase (DE: 199 43 587.1, DSM12968),
• The lysA- coding for the diaminopimelate decarboxylase Gen (Genbank entry accession number X07563; Yeh et al. (1988), Molecular and General Genetics 212: 112-119),
• The ddh coding for diaminopimelate dehydrogenase Gen (Ishino et al. (1988), Agricultural and Biological Chemistry 52 (11): 2903-2909)
• - The lysE gene encoding lysine export (DE-A-195 48 222),
• - The pyc gene coding for pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• - at the same time as the malate quinone oxidoreductase coding mqo gene (Molenaar et al. (1998), European Journal of Biochemistry 254: 395-403),
• - that for the zwa1 gene (DE: 199 59 328.0, DSM 13115),
• The gdh gene coding for glutamate dehydrogenase (Börmann et al (1992), Molecular Microbiology 6, 317-326)

simultaneously amplified, in particular overexpressed or be amplified. The simultaneous reinforcement of one or multiple genes selected from the group dapD, dapE and dapE is preferred.  

Furthermore, it may be advantageous for the production of L-lysine, in addition to the amplification of the dapC gene, optionally in combination with one or more genes selected from the group dapD, dapE and dapF, simultaneously

• - The coding for the phosphoenolpyruvate carboxykinase pck gene (DE 199 50 409.1, DSM 13047), or
• The pgj coding for the glucose-6-phosphate isomerase Gen (US 09 / 396,478, DSM 12969), or
• - The poxB gene coding for the pyruvate oxidase (DE: 199 51 975.7, DSM 13114), or
• The zwa2 gene (DE: 199 59 327.2, DSM 13113), or
• The genes coding for the succinyl-CoA synthetase sucC or sucD (DE: 199 56 686.0)

mitigate.

Furthermore, it can be used for the production of L-lysine, be beneficial, in addition to enhancing the dapC gene, optionally in combination with one or more genes selected from the group dapD, dapE and dapF, unwanted Eliminate side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention can continuous or discontinuous in the batch process (Batch cultivation) or in the fed batch (feed method) or repeated fed batch process (repetitive feed process) for the production of L-lysine. A summary of known cultivation methods are in the textbook of Chmiel (Bioprozesstechnik 1. Introduction into bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (Bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).  

The culture medium to be used must suitably Claims of the respective strains suffice. descriptions of culture media of various microorganisms are in Manual of Methods for General Bacteriology American Society for Bacteriology (Washington D.C., USA, 1981). As a carbon source can sugar and Carbohydrates such. Glucose, sucrose, lactose, Fructose, maltose, molasses, starch and cellulose, oils and Fats such as Soybean oil, sunflower oil, peanut oil and Coconut oil, fatty acids such. For example, palmitic acid, stearic acid and linoleic acid, alcohols such as. As glycerol and ethanol and organic acids such. As acetic acid can be used. These substances can be used singly or as a mixture become. As a nitrogen source, organic nitrogen containing compounds such as peptones, yeast extract, Meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate and ammonium nitrate can be used. The Nitrogen sources may be singly or as a mixture be used. As phosphorus source can phosphoric acid, Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used. The culture medium must further contain salts of metals such as As magnesium sulfate or iron sulfate, for the Growth are necessary. Finally, essential Growth substances such as amino acids and vitamins in addition to the above mentioned substances. The culture medium In addition, suitable precursors may be added. The mentioned feedstocks can be used to culture in the form of a added one-off approach or appropriately be fed during cultivation.

For pH control of the culture basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or Ammonia water or acidic compounds such as phosphoric acid or sulfuric acid used in a suitable manner. to Foam processing control can use anti-foaming agents z. B. fatty acid polyglycol esters are used. to  Maintaining the stability of plasmids can the Medium suitable selective substances such. B. Antibiotics are added. To aerobic conditions maintain, become oxygen or oxygen containing gas mixtures such. B. air into the culture entered. The temperature of the culture is usually at 20 ° C to 45 ° C, and preferably at 25 ° C to 40 ° C. The Culture is continued until a maximum is reached Lysine has formed. This goal is usually within 10 hours to 160 hours.

The analysis of L-lysine can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190). The following microorganism has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• Corynebacterium glutamicum strain DSM5715 / pXT-dapCexp as DSM 13254.

The inventive method is used for fermentative Production of L-lysine.  

Examples

The present invention will be described below with reference to Embodiments explained in more detail.

example 1 Preparation of a Genomic Cosmid Gene Bank Corynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33: 168-179) isolated and partially digested with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, Code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences USA 84: 2160-2164), obtained from the company Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, Code no. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. Subsequently, the cosmid DNA was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, Code no. 27-0868-04). The cosmid DNA treated in this way was mixed with the treated ATCC 13032 DNA and the mixture was mixed with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no. 27-0870-04). treated. The ligation mixture was then packaged in phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). To infect the E. coli strain NM554 (Raleigh et al 1988, Nucleic Acid Research 16: 1563-1575), the cells were taken up in 10 mM MgSO ₄ and mixed with an aliquot of the phage suspension. Infection and titering of the cosmidbank were performed as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) with 100 mg / L ampicillin. After incubation overnight at 37 ° C, recombinant single clones were selected.

Example 2 Isolation and sequencing of the dape gene

The cosmid DNA of a single colony was treated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to manufacturer's instructions isolated and with the Restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27-0913-02) partially split. The DNA fragments were with shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product no. 1758250) dephosphorylated. After gel electrophoresis Separation was the isolation of the cosmid fragments in Size range from 1500 to 2000 bp with the QiaExII gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pZero-1 related from Invitrogen (Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04) split. The Ligation of Cosmidfragmente in the Sequencing vector pZero-1 was prepared as described by Sambrook et al. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor) described, wherein the DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) Was incubated overnight. This ligation mixture was into the E. coli strain DH5αMCR (Grant, 1990,  Proceedings of the National Academy of Sciences U.S.A., 87: 4645-4649) (Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-7) and on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l Zeocin plated. The Plasmid preparation of the recombinant clones was performed with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out after the dideoxy Chain termination method of Sanger et al. (1977, Proceedings of the National Academies of Sciences U.S.A., 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). It became the "RR dRhodamin Terminator Cycle Sequencing Kit "by PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) used. Gel electrophoretic separation and Analysis of the sequencing reaction was carried out in one "Rotiphoresis NF Acrylamide / Bisacrylamide" Gel (29: 1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI Prism 377 "sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The obtained crude sequence data were then under Application of the Staden Program Package (1986, Nucleic Acids Research, 14: 217-231), version 97-0. The Single sequences of pZero1 derivatives became one coherent contig assembled. The computer-aided Coding area analysis was carried out with the program XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analyzes were carried out with the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25: 3389- 3402), against the non-redundant database of the "National Center for Biotechnology Information "(NCBI, Bethesda, MD, USA).

The resulting nucleotide sequence of the dapC gene is shown in SEQ ID No. 1 shown. Analysis of the nucleotide sequence revealed an open reading frame of 1101 base pairs, which is called dapC gene was designated. The dape gene codes for one A polypeptide of 367 amino acids, which is shown in SEQ ID no. 2 is shown.  

Example 3 Production of a shuttle vector pXT-dapCexp for Enhancement of the dapC gene in C. glutamicum 3.1. Cloning of the dapC gene

From strain ATCC 13032 according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) isolated chromosomal DNA. Because of the sequence of the dapC gene known from Example 2 for C. glutamicum, the following oligonucleotides were selected for the polymerase chain reaction:
DapC (dCex1):
5 'GAT CTA (GAA TTC) GCC TCA GGC ATA ATC TAA CG 3'
DapC (dCexna2):
5 'GAT CTA (TCT AGA) CAG AGG ACA AGG CAA TCG GA 3'

The primers shown were from the company ARK Scientific GmbH Biosystems (Darmstadt, Germany) synthesized and according to the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press) with Pwo polymerase from Roche Diagnostics GmbH (Mannheim, Germany) the PCR reaction carried out. With the help of the polymerase chain reaction allow the primers amplification of about 1.6 kb large DNA fragment carrying the dapC gene. also contain the primer DapC (dCex1) the sequence for the Interface of the restriction endonuclease EcoRI, and the Primer DapC (dCexna2) the interface of Restriction endonuclease XbaI described in the above marked nucleotide sequence marked by parentheses are.

The amplified DNA fragment of about 1.6 kb, which carries the dapC gene, was amplified with the Zero Blunt ™ kit from Invitrogen Corporation (Carlsbad, CA, USA, catalog number K2700-20) into the vector pCR®Blunt II (FIG. Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)). Subsequently, the E. coli strain Top10 was transformed with the ligation batch according to the kit manufacturer (Invitrogen Corporation, Carlsbad, CA, USA). The selection of plasmid-carrying cells was made by plating out the transformation batch on LB agar (Sambrook et al, Molecular cloning. A laboratory manual 2 ^nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989..), The 25 mg / l kanamycin had been supplemented. Plasmid DNA was isolated from a transformant using the QIAprep Spin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatment with the restriction enzyme XbaI and EcoRI with subsequent agarose gel electrophoresis (0.8%). The DNA sequence of the amplified DNA fragment was checked by sequencing. The plasmid was named pCRdapC. The strain was named E. coli Top10 / pCRdapC.

3.2. Production of the E. coli - C. glutamicum shuttle Vector pEC-XT99A

As the starting vector for the construction of the E. coli C. glutamicum shuttle expression vector pEC-XT99A became the E.coli expression vector pTRC99A (Amann et al., 1988, Gene 69: 301-315). After BspHI restriction cleavage (Roche Diagnostics GmbH, Mannheim, Germany, Product description BspHI, Product No. 1467123) and subsequent Klenow treatment (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01; Method according to Sambrook et al., 1989, Molecular Cloning: A Labor Manual, Cold Spring Harbor) was the Ampicillin residual gene (bla) against the tetracycline Resistance gene of the C. glutamicum plasmid pAG1 (GenBank Accession No. AF121000). This was the Resistance gene-bearing region as AluI fragment (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description AluI, Product No. 27-0884-01) in the  linearized E. coli expression vector pTRC99A cloned. The ligation was performed as described by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor) described, wherein the DNA mixture with T4 Ligase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description T4 DNA Ligase, Product No. 27-0870-04) incubated overnight. This ligation mixture was into the E. coli strain DH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87: 4645-4649) (Tauch et al., 1994, FEMS Microbiology Letters, 123: 343-7). The constructed E. coli Expression vector was designated pXT99A.

As a basis for cloning a minimal replica Corynebacterium glutamicum was the plasmid pGA1 (Sonnen et al. 1991, Gene, 107: 69-74). By BalI / PstI Restriction cleavage (Promega GmbH, Mannheim, Germany, Product Description BalI, Product No. R6691; Amersham Pharmacia Biotech, Freiburg, Germany Product description PstI, Product No. 27-0976-01) of the Vector pGA1 could a 3484 bp fragment in the with SmaI and PstI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description SmaI, Product No. 27-0942-02, Product description Pstl, Product No. 27-0976-01) fragmented vector pK18mob2 (Tauch et al., 1998, Archives of Microbiology 169: 303-312). Using BamHI / XhoI restriction cleavage (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description BamHI, Product No. 27-086803, Product Description XhoI, Product No. 27-0950-01) and subsequent Klenow treatment (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01; Method according to Sambrook et al., 1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor) an 839 bp fragment was deleted. From the T4 Ligase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description T4 DNA Ligase, Product No. 27-0870-04) religated construct could be the C. glutamicum Minimal replicon as a 2645 bp fragment in the E. coli  Expression vector pXT99A be cloned. For this purpose, the DNA of minimal replicon-bearing construct with the Restriction enzymes KpnI (Amersham Pharmacia Biotech, Freiburg, Germany, Product description KpnI, Product No. 27-0908-01) and PstI (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description PstI, Product No. 27-0886-03) and then using Klenow Polymerase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01) a 3'-5'- Exonuclease treatment (Sambrook et al., 1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor) carried out.

In a parallel approach, the E. coli Expression vector pXT99A with the restriction enzyme RsrII (Roche Diagnostics, Mannheim, Germany, Product description RsrII, Product No. 1292587) and with Klenow polymerase (Amersham Pharmacia Biotech, Freiburg, Germany, Klenow Fragment of DNA Polymerase I, Product No. 27-0928-01) for ligation. The Ligation of the minimal replica with the vector construct pXT99A was purified as described by Sambrook et al. (1989, Molecular Cloning: A laboratory manual, Cold Spring Harbor) described, wherein the DNA mixture with T4 Ligase (Amersham Pharmacia Biotech, Freiburg, Germany, Product Description T4 DNA Ligase, Product No. 27-0870-04) incubated overnight.

The thus constructed E. coli C. glutamicum shuttle Expression vector pEC-XT99A was electroporated (Liebl et al., 1989, FEMS Microbiology Letters, 53: 299-303) in C. glutamicum DSM5715. The selection of Transformants were on LBHIS agar consisting of 18.5 g / l Brain-Heart Infusion Boullion, 0.5 M sorbitol, 5 g / l Bacto-Tryptone, 2.5 g / L Bacto-Yeast extract, 5 g / L NaCl and 18 g / L Bacto agar containing 5 mg / L tetracycline had been supplemented. The incubation took place for 2 Days at 33 ° C.  

Plasmid DNA was made from a transformant according to the usual Isolated methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), with the Cut restriction endonuclease HindIII and the Plasmid by subsequent agarose gel electrophoresis checked.

The resulting plasmid construct was named pEC-XT99A and is shown in FIG . The strain obtained by electroporation of the plasmid pEC-XT99A into the Corynebacterium glutamicum strain DSM5715 was called DSM5715 / pEC-XT99A and deposited as DSM12967 in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty.

3.3. Cloning of dapC in E. coli C. glutamicum shuttle Vector pEC-XT99A

The vector used was the E. coli described in Example 3.2. C. glutamicum shuttle vector pEC-XT99A used. DNA This plasmid was digested with the restriction enzymes EcoRI and XbaI completely split and then shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250) dephosphorylated.

From that in example 3.1. described plasmid pCRdapC was the dapC gene by complete cleavage with the Enzymes EcoRI and XbaI isolated. The approx. 1600 bp big dapC Fragment was taken from the agarose gel with the QiaExII gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany) isolated.

The dapC fragment obtained in this way was mixed with the prepared vector pEC-XT99A and the mixture was mixed with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code no. 27-0870-04). treated. The ligation mixture was transformed into E. coli strain DH5α (Hanahan, In: DNA cloning, A practical approach, Vol. I. IRL-Press, Oxford, Washington DC, USA). The selection of plasmid-carrying cells was carried out by plating out the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 5 mg / l tetracycline. After incubation overnight at 37 ° C, recombinant single clones were selected. Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and cleaved with the restriction enzymes EcoRI and XbaI to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was named pXT-dapCexp. It is shown in FIG. 2.

Example 4 Transformation of strain DSM5715 with plasmid pXT- dapCexp

The strain DSM5715 was subjected to the plasmid pXT-dapCexp Application of the von Liebl et al., (FEMS Microbiology Letters, 53: 299-303 (1989)) transformed. The selection of the transformants was carried out on LBHIS agar consisting of 18.5 g / l Brain-Heart infusion Boullion, 0.5 M sorbitol, 5 g / l Bacto tryptone, 2.5 g / l Bacto-Yeast extract, 5 g / l NaCl and 18 g / l Bacto agar, the was supplemented with 5 mg / l tetracycline. The Incubation was for 2 days at 33 ° C.

Plasmid DNA was made from a transformant according to the usual Isolated methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), with the Restriction endonucleases EcoRI and XbaI cut and the plasmid by subsequent agarose gel electrophoresis checked. The resulting strain was DSM5715 / pXT-dapCexp named and as DSM 13254 at the German collection for Microorganisms and cell cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty.  

Example 5 Production of lysine

The C. glutamicum strain obtained in Example 4 DSM5715 / pXT-dapCexp was in production for lysine cultivated suitable nutrient medium and the lysine content in the Culture supernatant determined.

For this, the strain was first on agar plate with the appropriate antibiotic (brain-heart agar with tetracycline (5 mg / l)) for 24 hours at 33 ° C incubated. Starting from of this agar plate culture was inoculated into a preculture (10 ml Medium in 100 ml Erlenmeyer flask). As a medium for the Preculture, the complete medium CgIII was used.

Medium Cg III NaCl 2.5 g / l Bacto-peptone 10 g / l Bacto-yeast extract 10 g / l Glucose (separately autoclaved) 2% (w / v)

The pH was adjusted to pH 7.4 set.

To this was added tetracycline (5 mg / L). The preculture was 16 hours at 33 ° C at 240 rpm on the shaker incubated. From this Vorkultur became a main culture seeded so that the initial OD (660 nm) of the major culture 0.1 was. For the main culture, the medium became mm used.  

Medium MM CSL (Corn Steep Liquor) 5 g / l MOPS (morpholinopropanesulfonic acid) 20 g / l Glucose (separately autoclaved) 50 g / l (NH ₄ ) ₂ SO ₄ 25 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ .2H ₂ O 10 mg / l FeSO ₉ .7 H ₂ O 10 mg / l MnSO ₄ .H ₂ O 5.0 mg / l Biotin (sterile filtered) 0.3 mg / l Thiamine.HCl (sterile filtered) 0.2 mg / l L-leucine (sterile filtered) 0.1 g / l CaCO ₃ 25 g / l

CSL, MOPS and saline were adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions were added, as well as the dry autoclaved CaCO ₃ .

The cultivation is carried out in 10 ml volume in a 100 ml Erlenmeyer flask with harassment. It was tetracycline (5 mg / l) added. The cultivation took place at 33 ° C and 80% Humidity.

After 72 hours, the OD became at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed was with an amino acid analyzer from Eppendorf-BioTronik  (Hamburg, Germany) by ion exchange chromatography and post-column derivatization with ninhydrin detection certainly.

Table 1 shows the result of the experiment.

Table 1

SEQUENCE LISTING

The following figures are attached:

Fig. 1 Map of the plasmid pEC-XT99A

Fig. 2 Map of the plasmid pXT-dapCexp

The abbreviations and designations used have the following meaning.
by: gene for controlling the copy number from pGA1
oriE: plasmid-encoded replication origin of E. coli
rep: Plasmid-encoded replication origin from C. glutamicum plasmid pGA1
Ptrc: trc promoter from pTRC99A
T1, T2: terminator regions 1 and 2 from pTRC99A
lacIq: repressor gene of the Lac operon
Tet: resistance gene for tetracycline
dapC: dapC gene of C. glutamicum
EcoRI: restriction enzyme EcoRI site
EcoRV: restriction enzyme EcoRV site
HindIII: restriction enzyme HindIII site
KpnI: restriction enzyme KpnI site
SalI: Site of the restriction enzyme SalI
SmaI: Interface of the restriction enzyme SmaI
NdeI: Interface of the restriction enzyme NdeI
BamHI: Interface of the restriction enzyme BamHI
NcoI: restriction enzyme NcoI site
XbaI: Restriction enzyme XbaI site
SacI: restriction enzyme SacI site

Claims (16)

An isolated polynucleotide containing a polynucleotide sequence selected from the group

• a) polynucleotide which is at least 70% identical to a polynucleotide coding for a polypeptide containing the amino acid sequence of SEQ ID NO 2,
• b) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO 2,
• c) polynucleotide which is complementary to the polynucleotides of a) or b), or
• d) polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequences of a), b) or c).

2. polynucleotides according to claim 1, the polynucleotide being a coryneform bacterium is replicable, preferably recombinant DNA. 3. polynucleotides according to claim 1, wherein the polynucleotide is an RNA. 4. polynucleotides according to claim 2, containing the nucleic acid sequence as in SEQ ID NO 1 shown. 5. Replicatable DNA according to claim 2 containing

• a) the nucleotide sequence shown in SEQ ID NO 1, or
• b) at least one sequence corresponding to the sequences (i) within the range of the degeneracy of the genetic code, or
• c) at least one sequence that hybridizes with the sequences complementary to the sequences (i) or (ii), and optionally
• d) functionally neutral sense mutations in (i).

6. Vector, containing the polynucleotide according to claim 1, in particular pXT-dapCexp, characterized by the reproduced in Fig. 2 restriction map, deposited under the name DSM 13254 in Corynebacterium glutamicum. 7. Coryneform bacteria serving as the host cell, carrying the Vector according to claim 6, or in which the zwa1 gene is amplified. 8. A process for the preparation of L-amino acids, in particular L-lysine, characterized in that one carries out the following steps:

• a) fermentation of the desired L-amino acid-producing bacteria in which at least the dapC gene is amplified,
• b) enrichment of the desired product in the medium or in the cells of the bacteria, and
• c) isolating the L-amino acid.

9. The method according to claim 8, characterized, that one uses bacteria, in which one additionally other genes of the biosynthetic pathway of the desired L-amino acid amplified.   10. The method according to claim 8, characterized, that one uses bacteria in which the Metabolic pathways are at least partially eliminated which reduce the formation of L-lysine. 11. The method according to one or more of claims 8 until 12, characterized, that one uses coryneform bacteria, the L-lysine produce. 12. The method according to claim 8, characterized in that one fermented for the production of L-lysine bacteria, in which in addition to the dapC gene simultaneously one or more of the genes selected from the group

• 1. 12.1 the lysC gene coding for a feedback-resistant aspartate kinase,
• 2. 12.2 the asd gene coding for the aspartate-semialdehyde dehydrogenase,
• 3. 12.3 the dapA gene coding for the dihydrodipicolinate synthase,
• 4. 12.4 the dapB gene coding for the dihydrodipicolinate reductase,
• 5. 12.5 the dapD gene coding for the tetrahydrodipicolinate succinylase,
• 6. 12.6 the dapE gene encoding N-succinyl-diaminopimelate desuccinylase,
• 7. 12.7 the dapE gene encoding diaminopimelate epimerase,
• 8. 12.8 the lysA gene encoding diaminopimelate decarboxylase,
• 9. 12.9 the ddh gene encoding diaminopimelate dehydrogenase,
• 10. 12.10 the lysE gene encoding lysine export,
• 11. 12.11 the pyc gene coding for pyruvate carboxylase,
• 12. 12.12 the mqo gene coding for malate quinone oxidoreductase,
• 13. 12.13 that for the zwa1 gene
• 14. 12.14 the gdh gene encoding glutamate dehydrogenase

simultaneously amplified, in particular overexpressed or amplified. 13. The method according to claim 8, characterized in that one fermented for the production of L-lysine bacteria, in which simultaneously one or more of the genes selected from the group

• 1. 13.1 the peck gene coding for the phosphoenolpyruvate carboxykinase,
• 2. 13.2 the pgi gene coding for the glucose-6-phosphate isomerase,
• 3. 13.3 the poxB gene coding for the puryvate oxidase,
• 4. 13.4 the zwa2 gene,
• 5. 13.5 the succinyl-CoA synthetase coding genes sucC or sucD

weakens. 14. Method according to one or more of the preceding Claims, characterized, that one microorganisms of the genus Corynebacterium glutamicum. 15. Use of polynucleotide sequences according to claim 1 as hybridization probes for the isolation of cDNA, the encoded for the dapC gene product. 16. Use of polynucleotide sequences according to claim 1 as hybridization probes for the isolation of cDNA or Genes that are very similar to the sequence of the have dapC gene.